Abstract
Systemic uncontrolled inflammatory response, also termed as sepsis, is responsible for many mortalities. Bacterial endotoxin, lipopolysaccharide (LPS), is a major cause of sepsis in endothelial cells. Even though a lot of research has been done to define underlying mechanisms of LPS induced sepsis, the role of long non-coding RNAs (lncRNAs), a group of >200 kb RNAs in sepsis is not well-defined. Expression of pro-inflammatory mediators IL6, ICAM1, and VCAM1 (which encodes interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, respectively) were determined following LPS treatment of human dermal microvascular endothelial cells (HMECs) for 24 h to confirm sepsis induction. RNA immunoprecipitation (RIP) analysis was performed using the chromatin modifying proteins (CMPs), heterogeneous nuclear ribonucleoprotein (hnRNP) K and corepressors of the RE-1 silencing transcription factor (coREST) as individual baits. Quantitative real time polymerase chain reaction (qRT-PCR) was performed on RNA isolated from immunoprecipitated pellets for six different lncRNAs. The effect of the differentially expressed lncRNAs were determined by ectopic overexpression of the lncRNAs before induction of sepsis. Expression of IL6, ICAM1, and VCAM1 were significantly upregulated following treatment of the HMECs with LPS for 24 h confirming induction of sepsis. RIP and qRT-PCR analysis revealed that the lncRNAs HULC, UCA1, and MALAT-1 were significantly enriched with the CMPs after sepsis. RNA interference using siRNAs targeting HULC and UCA1, but not MALAT-1, decreased the expression of IL6, ICAM1, and VCAM1 to endogenous levels. Our results were further validated in an in vivo model of sub-lethal LPS-induced sepsis, whereby siRNA mediated knockdown of UCA1 and HULC lncRNAs prevented induction of VCAM1, ICAM1, and IL6, as assayed by immunohistochemistry. Cumulatively, these results suggest that LPS induced in vitro sepsis in endothelial cells and induction of pre-inflammatory mediators are at least in part due to increased expression of the UCA1 and HULC lncRNAs.
Highlights
Sepsis is an uncontrolled systemic inflammatory response that can affect multiple organs in the body and is a major cause of worldwide mortality (Dauphinee and Karsan, 2006; Vincent et al, 2011; Randolph and McCulloh, 2014)
Human dermal microvascular endothelial cells were treated with LPS for 24 h and RNA isolated from untreated and LPS treated human dermal microvascular endothelial cells (HMECs) were assayed for relative expression of the pro-inflammatory mediators, ICAM1, VCAM1, and IL6
Silencing of HULC significantly decreased the expression of ICAM1, VCAM1, and IL6 in the LPS treated HMECs to those observed in the endogenous untreated HMECs (ICAM1 14.2 ± 1.9 folds in LPS group vs. 2.1 ± 0.04 in LPS + si-HULC group, P < 0.05; VCAM1: 9.3 ± 1.09 folds in LPS group vs. 2.3 ± 0.02 in LPS + si-HULC group, P < 0.05; and, IL6: 10.1 ± 0.9 folds in LPS group vs. 1.86 ± 0.07 in LPS + si-HULC group, P < 0.05) (Figures 4C, 6). These results indicate that increase in proinflammatory ICAM1, VCAM1, and IL6 in HMECs during LPS induced sepsis in vitro is at least in part due to increase in expression of the long non-coding RNAs (lncRNAs) HULC and urothelial carcinoma-associated 1 (UCA1)
Summary
Sepsis is an uncontrolled systemic inflammatory response that can affect multiple organs in the body and is a major cause of worldwide mortality (Dauphinee and Karsan, 2006; Vincent et al, 2011; Randolph and McCulloh, 2014). When lipopolysaccharide (LPS), present within the cell wall of Gram-negative bacteria, encounters endothelial cells, a signaling cascade is induced resulting in sepsis (Dauphinee and Karsan, 2006) in initiated. Endothelial cells line the microcirculatory route, maintaining an antithrombotic surface and in turn regulating vasomotor tonicity and normal blood flow (Danese et al, 2007; Chelazzi et al, 2015). Exposure of the microvascular endothelium to inflammatory cytokines or to endotoxins like LPS result in induced expression of pro-inflammatory markers inclusive of interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) (Danese et al, 2007; Chelazzi et al, 2015). Given the central role of LPS in mediating sepsis and septic shock it is not surprising that the mechanisms regulating the pathophysiology of sepsis has been studied in detail (Beck et al, 2006; Singh et al, 2016; Yu et al, 2017)
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